Guo Zhongxin, Wang Xian-Bing, Li Wan-Xiang, Ding Shou-Wei
Vector-Borne Virus Research Center, Haixia Institute of Science and Technology, College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, Fujian, People's Republic of China.
State Key Laboratory of Agro-Biotechnology, College of Biological Sciences, China Agricultural University, Beijing, People's Republic of China.
Methods Mol Biol. 2019;2028:215-229. doi: 10.1007/978-1-4939-9635-3_12.
RNA interference (RNAi) acts as a natural defense mechanism against virus infection in plants and animals. Much is known about the antiviral function of the core RNAi pathway components identified mostly by genetic screens based on specific RNAi of cellular mRNAs. Here we describe a sensitized genetic screening system for the identification of novel components and regulators in the antiviral RNAi pathway established in the model plant species Arabidopsis thaliana. Our genetic screen identifies antiviral RNAi (avi)-defective Arabidopsis mutants that develop visible disease symptoms after infection with CMV-∆2b, a Cucumber mosaic virus mutant deficient in the expression of its viral suppressor of RNAi. Loss of RNAi suppression renders CMV-∆2b highly susceptible to antiviral RNAi so that it replicates to high levels and induces disease development only in avi mutants. This chapter provides the methods for the propagation of CMV-∆2b, preparation of the mutant plants for virus inoculation, identification and characterization of avi mutants, and cloning of the genes responsible for the mutant phenotype by either the genetic linkage to T-DNA insertion or a mapping-by-sequencing approach.
RNA干扰（RNAi）作为植物和动物抵御病毒感染的一种天然防御机制。基于细胞mRNA的特异性RNAi，通过遗传筛选大多已鉴定出核心RNAi途径成分的抗病毒功能，对此我们已了解很多。在此，我们描述了一种用于鉴定模式植物拟南芥中抗病毒RNAi途径新成分和调控因子的敏感遗传筛选系统。我们的遗传筛选鉴定出抗病毒RNAi（avi）缺陷型拟南芥突变体，这些突变体在感染黄瓜花叶病毒突变体CMV-∆2b后会出现明显的病害症状，CMV-∆2b是一种其RNAi病毒抑制子表达缺失的黄瓜花叶病毒突变体。RNAi抑制作用的丧失使CMV-∆2b极易受到抗病毒RNAi的影响，从而使其大量复制，并仅在avi突变体中诱导病害发展。本章提供了CMV-∆2b繁殖、用于病毒接种的突变体植物制备、avi突变体的鉴定和表征，以及通过与T-DNA插入的遗传连锁或测序定位方法克隆导致突变体表型的基因的方法。
